Growing evidence points to a critical role for epigenetic mechanisms in diverse aspects of human health and disease, including neurodegenerative and neuropsychiatric disorders. This role includes fundamental cellular processes ranging from neurogenesis to synaptogenesis, with significant implications for the development of novel therapeutics to treat and ideally prevent disease pathophysiology. However, despite dramatic advances in our ability to observe the epigenome and transcriptome, our ability to perturb the epigenome and manipulate transcriptional programs with precise temporal control and spatial resolution remains severely limited due to the pleiotropic effects of most existing pharmacological probes and the lack of suitable genetic tools. To overcome these limitations and enable targeting specific cell types within neurocircuits, we propose an integrated, multidisciplinary approach-spanning synthetic chemistry to neurobiology- combining innovative, and scalable 'chemical optoepigenetic' technologies together with epigenome and transcriptome analysis in human and mouse neurons. Our strategy for neuromodulation exploits photoswitchable compounds with fast thermal relaxation kinetics that possess slow-binding kinetics with their epigenetic targets. Using the family of class I histone deacetylase (HDAC)-containing chromatin-modifying complexes, which our work has demonstrated as key regulators of chromatin-mediated neuroplasticity, to advance the testing of this methodology, the specific aims of the proposed project are to: 1) synthesize, characterize and optimize the physical properties, biochemical potency and selectivity of optoepigenetic probes capable of light-dependent inhibition of the deacetylase activity of neuronal chromatin-modifying complexes containing different class I HDAC isoforms; 2) determine the epigenome and transcriptome changes in cultured human stem cell-derived neurons after precise temporal manipulation of different HDAC complexes; and 3) use the novel optoepigenetic probes to temporally manipulate the epigenome of spatially defined mouse neurons to enhance synaptogenesis and modulate hippocampal circuit function. Overall, by providing significant improvements in spatiotemporal control of HDAC activity in combination with advances in the generation of isoform and complex-selective HDAC inhibitors, we anticipate our approach will limit the pleiotropic effects of currently available small molecule tools. Through selective manipulation of the epigenome in specific regions of neurocircuits, we anticipate being able to significantly improve our understanding of how specific temporal regulation of epigenetic states affects neuroplasticity and to be able to delineate the contribution of epigenetic mechanisms in defined neuronal subtypes within neurocircuits. Importantly, our approach developing chemical optoepigenetic probes is broadly applicable to manipulating epigenetic regulatory mechanisms and could be scaled to enable the assembly of a molecular tool kit for combinatorial optoepigenetic studies. Such tools could have wide applicability in the field of neuroepigenetics and help advance efforts to develop improved therapeutics targeting neuroplasticity.